The invention comprises protein single chain variants of tissue type plasminogen activator, also referred to as t-PA as well as nucleic acids encoding such protein single chain variants of tissue type plasminogen activator. The t-PA protein variants have higher zymogenicity than the wild-type single chain t-PA form. Methods of making and using the t-PA variant compositions are also described.
Tissue-type plasminogen activator (t-PA) is a serine protease that plays a critical role in the process of fibrinolysis, the dissolution of clots, by activating plasminogen to the protease plasmin. t-PA has been fully identified and characterized by underlying DNA sequence and deduced amino acid sequence. See Pennica et al., Nature, 301: 214 (1983) and U.S. Pat. No. 4,853,330, issued Aug. 1, 1989, the teachings of both of which are incorporated by reference. The nucleotide sequence and deduced primary amino acid sequence of human t-PA is depicted in FIG. 1A, FIG. 1B and FIG. 1C.
The group of amino acid residues from xe2x88x9235 to xe2x88x921 preceding the sequence of the mature t-PA is the xe2x80x9cproxe2x80x9d sequence. The mature t-PA molecule (amino acid residues 1-527) contains five domains that have been defined with reference to homologous or otherwise similar structures identified in various other proteins such as trypsin, chymotrypsin, plasminogen, prothrombin, fibronectin, and epidermal growth factor (EGF). These domains have been designated, starting at the N-terminus of the amino acid sequence of mature t-PA, as 1) the finger region (F) that has variously been defined as including amino acid residues 1 to about 44, 2) the growth factor region (G) that has been variously defined as stretching from about amino acid residues 45 to 91 (based upon its homology with EGF), 3) kringle one (K1) that has been defined as stretching from about amino acid residue 92 to about amino acid residue 173, 4) kringle two (K2) that has been defined as stretching from about amino acid residue 180 to about amino acid residue 261, and 5) the so-called serine protease domain (P) that generally has been defined as stretching from about amino acid residue 264 to the C-terminal end of the molecule at amino acid residue 527. These domains, which are situated generally adjacent to one another, or are separated by short xe2x80x9clinkerxe2x80x9d regions, account for the entire amino acid sequence of from 1 to 527 amino acid residues of the mature form of t-PA.
Each domain has been described variously as contributing certain specific biologically significant properties. The finger domain has been characterized as containing a sequence of at least major importance for high binding affinity to fibrin. (This activity is thought important for the high specificity that t-PA displays with respect to clot lysis at the locus of a fibrin-rich thrombus.) The growth factor-like region likewise has been associated with cell surface binding activity. The kringle 2 region also has been strongly associated with fibrin binding and with the ability of fibrin to stimulate the activity of t-PA. The serine protease domain is responsible for the enzymatic cleavage of plasminogen to produce plasmin.
t-PA is unusual among proteases in the level of the enzymatic activity of its precursor. In general, proteases are synthesized as zymogens, inactive precursors that must either be proteolytically processed or bind to a specific co-factor to develop substantial catalytic activity. The increase in catalytic efficiency after zymogen activation, or zymogenicity, is dramatic in almost all cases, although varying widely among individual members of the chymotrypsin family. For example, strong zymogens, i.e., those having high zymogenicity, such as trypsinogen, chymotrypsinogen, or plasminogen are almost completely inactive, with measured zymogenicities of 104 to 106 (Robinson, N. C., Neurath, H., and Walsh, K. A. (1973) Biochemistry 12, 420-426; Gertler, A., Walsh, K. A., and Neurath, H. (1974) Biochemistry 13, 1302-1310). Other serine proteases exhibit intermediate zymogenicity. For example, the enzymatic activity of Factor XIIa is 4000-fold greater than that of its corresponding zymogen, Factor XII (Silverberg, M., and Kaplan, A. P. (1982) Blood 60, 64), and the catalytic efficiency of urokinase is 250-fold greater than that of pro-urokinase (Lijnen, H. R., Van Hoef, B., Nelles, L., and Collen, D. (1990) J. Biol. Chem. 265, 5232-5236). By contrast, the catalytic activities of single and two chain t-PA vary by a factor of only 5-10.
The zymogenicity, expressed as the ratio of the activity of the mature cleaved two-chain enzyme to that of the single chain precursor form, is only 5-10 for wild-type t-PA, in contrast to other precursors of other proteases that have little or no catalytic activity. Thus, the single chain form of wild-type t-PA is not a true zymogen.
There have been many attempts to improve the usefulness of t-PA by genetic engineering. These efforts have been only partially successful. The state of the art has been reviewed by Krause, J., and Tanswell, P. Arzneim.-Forsch. 39: 632-637 (1989) and in U.S. Pat. No. 5,616,486, the teachings of both of which are incorporated by reference.
Despite the profound advantages associated with natural t-PA as a clot-dissolving agent, it is not believed that the natural protein necessarily represents the optimal t-PA agent under all circumstances. Therefore, several variants have been proposed or devised to enhance specific properties of t-PA. Certain of those variants address disadvantages associated with the use of natural t-PA in situations where an agent with a longer half-life or slower clearance rate would be preferred, e.g., in the treatment of deep-vein thrombosis and following reperfusion of an infarct victim, or where a single-chain agent is preferred.
For example, removal of a substantial portion or all of the finger domain results in a molecule with substantially diminished fibrin binding characteristics, albeit in return there is a decrease in the overall rate of clearance of the resultant entityxe2x80x94See WO 89/00197 published Jan. 12, 1989.
Variants are described in EPO Pat. Publ. No. 199,574 that have amino acid substitutions at the proteolytic cleavage sites at positions 275, 276, and 277. These variants, characterized preferentially as t-PA variants having an amino acid other than arginine at position 275, are referred to as protease-resistant one-chain t-PA variants in that, unlike natural t-PA, which can exist in either a one-chain or two-chain form, they are resistant to protease cleavage at position 275 and are therefore not converted metabolically in vivo into a two-chain form. This form is thought to have certain advantages biologically and commercially, in that it is more stable while the fibrin binding and fibrin stimulation are increased relative to two-chain t-PA. Furthermore, plasminogen activators are described that comprise one domain capable of interacting with fibrin and the protease domain of urokinase, with one embodiment being urokinase altered to make it less susceptible to forming two-chain urokinase. See WO 88/05081 published Jul. 14, 1988.
For further patent literature regarding modification of the protease cleavage site of t-PA, see, for example, EPO Pat. Nos. 241,209; EP 201,153 published Nov. 12, 1986; EP 233,013 published Aug. 19, 1987; EP 292,009 published Nov. 23, 1988, EP 293,936 published Dec. 7, 1988; and EP 293,934 published Dec. 7, 1988; and WO 88/10119.
Glycosylation mutants at positions 117-119, 184-186, and 448-450 exhibited higher specific activity as the mole percent carbohydrate was reduced. See EPO Pub. No. 227,462 published Jul. 1, 1987. This patent application additionally discloses using an assay of fibrin/fibrin degradation products and teaches that one may also modify the t-PA molecule at positions 272-280 or delete up to 25 amino acids from the C-terminus. Further, the t-PA mutants with Asn 119, Ala 186 and Asn 450, which have the N-glycosylation sites selectively removed by DNA modification but contain residual O-linked carbohydrate, were found to be about two-fold as potent as melanoma t-PA in an in vitro lysis assay. See EPO Publ. No. 225,286 published Jun. 10, 1987.
Replacement of the amino acid at position 449 of t-PA with any amino acid except arginine to modify the glycosylation site, as well as modification of Arg 275 or deletion of the xe2x88x923 to 91 region, is also taught. See WO 87/04722 published Aug. 13, 1987. An amino acid substitution at position 448 of t-PA is disclosed as desirable to remove glycosylation. See EPO Pub. No. 297,066 published Dec. 28, 1988. The combination of modifications at positions 448-450 and deletion of the N-terminal 1-82 amino acids is disclosed by WO 89/00191 published Jan. 12, 1989. Additionally, urokinase has been modified in the region of Asp 302-Ser 303-Thr 304 to prevent glycosylation. See EPO Pub. No. 299,706 published Jan. 18, 1989.
However, alteration of the glycosylation sites, and in particular that at amino acid 117, seems invariably to result in a molecule having affected solubility characteristics that may result additionally in an altered circulating half-life pattern and/or fibrin binding characteristics. See EPO Pat. Publ. No. 238,304, published Sep. 23, 1987.
When the growth factor domain of t-PA is deleted, the resultant variant is still active and binds to fibrin, as reported by A. J. van Zonneveld et al., Thrombos. Haemostas. 54 (1): 4 (1985). Various deletions in the growth factor domain have also been reported in the patent literature. See EPO Publ. No. 241,209 (del-51-87), EPO Publ. No. 241,208 (del-51-87 and del-51-173), PCT 87/04722 (deletion of all or part of the N-terminal 1-91), EPO Publ. No. 231,624 (all of growth factor domain delexed), and EPO Publ. No. 242,830 and Jap. Pat. Appl. Kokai No. 62-269688 (some or all of the growth factor domain deleted).
It has further been shown tat t-PA can be modified both in the region of the first kringle domain and in the growth factor domain, resulting in increased circulatory half-life. See EPO Pat. Publ. No. 241,208 published Oct. 14, 1,987. The region between amino acids 51 and 87, inclusive, can be deleted from t-PA to result in a variant having slower clearance from plasma. Browne et al., J. Biol. Chem., 263; 1599-1602 (1988). Also, t-PA can be modified, without adverse biological effects, in the region of amino acids 67 to 69 of the mature, native t-PA, by deletion of certain amino acid residues or replacement of one or more amino acids with different amino acids. See EPO Pat. Publ. No. 240,334 published Oct. 7, 1987.
A hybrid of t-PA/urokinase using the region of t-PA encompassing amino acids 273-527 is also disclosed. See EPO 290,118 published Nov. 9, 1988. Serpin-resistant mutants of human t-PA with alterations in the protease domain, including de1296-302 t-PA, R304S t-PA, and R304E t-PA, are disclosed in Madison et al., Nature, 339: 721-724 (1989). The above list is not an exhaustive review of the numerous variants of t-PA that have been described.
As a result of the catalytic activity of precursor t-PA, despite effective clot lysis at targeted sites, nondesirable proteolysis occurs systemically resulting in the deleterious depletion of circulating fibrinogen, xcex12-anti-plasmin and plasminogen. What is needed are more zymogenic t-PA variant proteins that provide effective local clot lysis with diminished systemic proteolytic effects.
The present invention provides single chain variant t-PA proteins having at least two substitutions of basic amino acid residues by neutral or acidic amino acid residues, compared to the wild-type human t-PA, as well as polynucleotides encoding such single chain variant t-PA proteins. The single chain variant t-PA proteins of the present invention have the R275 amino acid residue substituted by an amino acid residue chosen from the group consisting of glycine, serine, threonine, asparagine, tyrosine, glutamine, aspartic acid, and glutamic acid. Preferably the single chain variant t-PA proteins of the present invention have the R275 amino acid residue substituted by an amino acid residue chosen from the group consisting of an aspartic acid residue and a glutamic acid residue, and most preferably by a glutamic acid residue.
The single chain variant t-PA proteins of the present invention have additionally at least one other basic amino acid residue in the serine protease region residue substituted by a non-basic amino acid such that the salt bridge interaction normally found in wildtype single chain t-PA between aspartate 477 and lysine 429 is disrupted. Preferably, basic amino acids are replaced with polar or acidic amino acids, and more preferably, amino acid residues chosen from the group consisting of glycine, serine, threonine, asparagine, tyrosine, glutamine, aspartic acid and glutamic acid.
The salt bridge interaction between aspartate 477 and lysine 429 can be disrupted by a substitution at position 477 or 429, or by an appropriate substitution at a position within the serine protease region that provides an alternative salt bridge interaction partner for at least one of aspartate 477 and lysine 429. In one preferred embodiment, the H417 amino acid residue is substituted by an amino acid residue chosen from the group consisting of glycine, serine, threonine, asparagine, tyrosine, glutamine, aspartic acid, and glutamic acid. More preferably the single chain variant t-PA proteins of the present invention have both the R275 amino acid residue and the H417 amino acid residue substituted by an amino acid residue chosen from the group consisting of an aspartic acid residue and a glutamic acid residue. Two exemplary preferred single chain variant t-PA proteins are the t-PA variants designated as R275E,H417E and R275E,H417D.
In another preferred embodiment, the K429 amino acid residue is substituted by an amino acid residue chosen from the group consisting of glycine, serine, threonine, asparagine, tyrosine, glutamine, aspartic acid, and glutamic acid. More preferably the single chain variant t-PA proteins of the present invention have both the R275 amino acid residue and the K429 amino acid residue substituted by an amino acid residue chosen from the group consisting of glycine, serine, threonine, asparagine, tyrosine, glutamine, aspartic acid, and glutamic acid. One preferred single chain variant t-PA protein is the t-PA variant designated as R275E,K429Y.
The single chain variant t-PA proteins of the present invention exhibit greater zymogenicity, expressed as the ratio of the activity of the mature cleaved two-chain enzyme to that of the single chain precursor form, than that of the wild type single chain t-PA protein. The single chain variant t-PA proteins of the present invention have zymogenicity of at least 10, preferably about 50 to about 200.
The single chain variant t-PA proteins of the present invention exhibit a greater fibrin stimulation factor, expressed as the ratio of the catalytic efficiencies in the presence and absence of fibrin, compared to the wild type single chain t-PA protein. The single chain variant t-PA proteins of the present invention have a fibrin stimulation factor of at least 7,000, preferably about 20,000 to about 50,000.
The single chain variant t-PA proteins of the present invention exhibit a reduced inhibition by plasminogen activator inhibitor 1 (PAI-1) to the wild type single chain t-PA protein. The single chain variant t-PA proteins of the present invention are at least a factor of 5, preferably at least a factor of about 9, most preferably at least a factor of about 200 less inhibited by PAI-1 compared to the wild type single chain t-PA protein.
The single chain variant t-PA proteins of the present invention exhibit a greater fibrin selectivity factor, expressed as the ratio of the catalytic efficiencies in the presence fibrin to that in the presence of fibrinogen, compared to the wild type single chain t-PA protein. Preferred embodiments of the single chain variant t-PA proteins of the present invention have a fibrin selectivity factor of at least 10, preferably at least 50, more preferably at least 100.